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PubMed1995

Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium.

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.

Animals↗

PubMed1984

Further characterization of Marek's disease virus-infected lymphocytes. II. In vitro infection.

Lymphocyte cultures from chicken spleens had been shown to be susceptible to in vitro infection by Marek's disease virus (MDV)4 in an earlier report from this laboratory. In that study, virus infection was evidenced by virus isolation and detection of viral internal antigen (VIA) 2 days post inoculation (DPI), and serial passage was accomplished by adding fresh spleen cells at 2-day intervals. The susceptible cells were identified as bursa-derived lymphocytes (B cells). Using a dual fluorescence technique to identify surface markers for B cells, thymus-derived lymphocytes (T cells) or Ia antigen on VIA-positive cells, we have now shown that a small proportion (generally less than 10%) of VIA-positive cells observed 2 DPI are T cells, and that a low level of infection can be maintained by serial passage of MDV in cultures totally free of B cells. Most infected T cells in this study had Ia antigen. As the incubation period for infected cultures was extended from 2 to 4 or 5 days, the average number of viable cells decreased but the percentage of viable cells infected with MDV (VIA-positive) increased. Also, both the proportion and the actual number of infected T cells increased, significantly more so in cultures from genetically susceptible P-2 donors than from resistant N-2 donors. Spleen-cell cultures from resistant Line 6 chickens were markedly less susceptible than those from susceptible Line 7 chickens to in vitro MDV infection, as assessed by numbers of VIA-positive cells at 5 DPI.

Animals↗

PubMed1976

Poxvirus infection of the baboon (Papio cynocephalus).

Ten serial passages of monkeypox (MPV), vaccinia, variola, and chimpanzeepox (chimp-9) viruses were performed in baboons (Papio cynocephalus) via skin scarification. Comparisons of clinical and virological results indicate that MPV and vaccinia are very closely related and that variola and chimp-9 viruses are identical. These findings suggest that infections of simians with chimp-9 virus resulted from contact with variola virus, the source of which is still unknown. On the other hand, MPV in monkeys may have resulted from contact with recently vaccinated humans, serial passage through simian hosts resulting in the biological alterations that produced MPV.

Animals↗

PubMed1967

Studies on persistent infections of tissue culture. VI. Reversible changes in Newcastle disease virus populations as a result of passage in L cells or chick embryos.

Populations of the Victoria strain of Newcastle disease virus (NDV), reisolated from persistently infected L-cell cultures and passed twice in the embryonated hen's egg (NDV(L-E-2)), were found to differ strikingly from the original, chick embryo-adapted virus (NDV(o)). After exposure of L cells to NDV(o) at high multiplicities of infection, all cells became abortively infected; they produced only small aggregates of viral antigen and few, if any, infectious virus particles, but they yielded large amounts of interferon. No cytopathic effects (CPE) were noted, and the cultures survived readily as viral carriers. In contrast, NDV(L-E-2) yielded under similar conditions large quantities of viral antigen and infectious virus particles, but no detectable interferon, and the cultures were rapidly destroyed. This change in "virulence" was at least partially reversible by further serial passages of NDV(L-E-2) in chick embryos, as was evident from a consecutive decrease in CPE with a concomitant increasingly rapid recovery of the L-cell cultures, gradually diminishing yields of infectious viral progeny, and the returning of a capacity to induce interferon synthesis. Thus, NDV(L-E-16) resembled NDV(o) in many aspects, except for a less striking reduction in its ability to replicate in L cells. Although a selection of viral variants under the given sets of conditions has not been entirely excluded, the establishment of "avirulence" appears to be largely explained by a gradual accumulation of noninfectious, interferon-inducing components in the course of serial passages in the embryonated hen's egg, and the acquisition of "virulence" by a loss of these components. The evidence is as follows. (i) By a step-wise decrease in the dose of virus and restriction of the analyses to the first infectious cycle, a multiplicity of infection was ultimately reached for all "avirulent" populations at which infected cells produced normal yields of infectious viral progeny; i.e., the interferon-inducing components were diluted to noneffective levels. The lowest multiplicity which resulted in a measurable reduction in infectious virus replication was also the last one to induce detectable interferon synthesis. (ii) All viral clones derived from "avirulent" populations behaved like NDV(L-E-2) rather than like the parent viral suspensions, except that some of them elicited small amounts of interferon in L cells. The interferon-inducing components were reduced or lost in the cloning procedures. The nature of the interferon-inducing components has not been established. These components, which were neutralized by rabbit sera against "virulent" NDV(L-E-2) populations, may represent largely inactive or incomplete virus particles; however, the infectious virus-hemagglutinin ratios of "avirulent" populations were mostly of an order similar to those of "virulent" populations. The interferon-inducing components aborted the infectious process in cells simultaneously invaded by infectious virus particles. The implications of these findings are discussed.

Animals↗

PubMed2005

Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro.

Our studies have shown that zeatin, (6-[4-hydroxy-3-methyl-but-2-enylamino]adenine), a cytokinin plant growth factor, has gerontomodulatory, youth preserving and anti-aging effects on serially passaged human adult skin fibroblasts undergoing aging in vitro. There were no immediate negative or toxic effects in terms of cell attachment, cell proliferation, cell survival, cytoskeletal organization, and cellular growth by treatment with zeatin concentrations between 1 and 200 microM. During long-term treatment, cells could be maintained throughout their replicative lifespan in the presence of 40, 80, and 200 microM zeatin, but the optimal concentration of zeatin's anti-aging and youth preserving effects was found to be 80 microM. Life-long serial passaging of human skin fibroblasts in the presence of zeatin resulted in the prevention of cell enlargement, reduction of intracellular debris, prevention of actin polymerization, and enhancement of cellular ability to decompose hydrogen peroxide and to cope with ethanol and oxidative stresses. Most importantly, anti-aging and beneficial effects of zeatin were observed without any induction of additional cell proliferation or an increase in the maximum proliferative capacity, thus ruling out any potentially harmful and carcinogenic effects.

Cellular Senescence↗

PubMed1982

Site-specific cleavage/packaging of herpes simplex virus DNA and the selective maturation of nucleocapsids containing full-length viral DNA.

Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets of the standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (M(r) 100 x 10(6)). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in M(r) from 5.5 to 100 x 10(6). These smaller DNA species were shown to consist of different integral numbers (from 1 to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (i) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (ii) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (iii) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm.

Capsid↗

PubMed1994

Temporal correlation between a single amino acid change in the VP4 of a porcine rotavirus and a marked change in pathogenicity.

We previously described a marked increase in the pathogenicity of a cell culture grown porcine rotavirus, PRV 4F, during serial passage in gnotobiotic piglets (Bridger et al., 1992). Here we report close temporal correlation between this change in pathogenicity and an amino acid change within a highly conserved hydrophobic domain of VP4 at position 469. Cell culture grown PRV 4F is unique in having a hydrophilic residue, glutamine, at amino acid 469; all previously sequenced VP4s have hydrophobic leucine or phenylalanine residues at the corresponding position. The detection of a point mutation causing a deduced amino acid change from glutamine to leucine at amino acid 469 of PRV 4F VP4 in virus obtained from one piglet at the second serial passage correlated exactly with the emergence of viral pathogenicity. However, given the multifactorial nature of virus pathogenicity, genetic studies are required to ascertain the degree to which this mutation is responsible for the observed change in pathogenicity.

Amino Acid Sequence↗

PubMed1980

Somatic cell hybrids of canine peritoneal macrophages and SV40-transformed human cells: derivation, characterization, and infection with Ehrlichia canis.

Somatic cell hybrids were obtained by fusion of canine peritoneal macrophages and SV40-transformed human skin fibroblasts. A cell line (WRH-2) was established from a single isolated hybrid clone. The WRH-2 cell line has been serially passaged 60 times and has a population doubling time of approximately 24 hours. Karyotypic analysis showed the modal number of chromosomes to be 80, with a selective segregation of canine chromosomes. Expression of incorporated canine DNA was substantiated by cellular enzyme activities and antigen expression. The susceptibility of 5% to 7% of WRH-2 cells to Ehrlichia canis infection was associated with phagocytic properties of these cells. Magnetic separation of phagocytic cells after ingestion of carbonyl iron resulted in a significant enhancement of the phagocytic population with a concomitant increase in the percentage of cells susceptible to ehrlichial infection. Serial passage of the selected subpopulation of hybird cells, however, resulted in a rapid diminution in the percentage of phagocytic cells.

Animals↗

PubMed1982

Structure and expression of class II defective herpes simplex virus genomes encoding infected cell polypeptide number 8.

Defective genomes present in serially passaged virus stocks derived from the tsLB2 mutant of herpes simplex virus type 1 were found to consist of repeat units in which sequences from the U(L) region, within map coordinates 0.356 and 0.429 of standard herpes simplex virus DNA, were covalently linked to sequences from the end of the S component. The major defective genome species consisted of repeat units which were 4.9 x 10(6) in molecular weight and contained a specific deletion within the U(L) segment. These tsLB2 defective genomes were stable through more than 35 sequential virus passages. The ratios of defective virus genomes to helper virus genomes present in different passages fluctuated in synchrony with the capacity of the passages to interfere with standard virus replication. Cells infected with passages enriched for defective genomes overproduced the infected cell polypeptide number 8, which had previously been mapped within the U(L) sequences present in the tsLB2 defective genomes. In contrast, the synthesis of most other infected cell polypeptides was delayed and reduced. The abundant synthesis of infected cell polypeptide number 8 followed the beta regulatory pattern, as evident from kinetic studies and from experiments in which cycloheximide, canavanine, and phosphonoacetate were used. However, in contrast to many beta (early) and gamma (late) viral polypeptides, the synthesis of infected cell polypeptide number 8 was only minimally reduced when cells infected with serially passaged tsLB2 were incubated at 39 degrees C. The tsLB2 mutation had previously been mapped within the domains of the gene encoding infected cell polypeptide number 4, the function of which was shown to be required for beta and gamma viral gene expression. It is thus possible that the tsLB2 mutation affects the synthesis of only a subset of the beta and gamma viral polypeptides. An additional polypeptide, 74.5 x 10(3) in molecular weight, was abundantly produced in cells infected with a number of tsLB2 passages. This polypeptide was most likely expressed from truncated gene templates within the most abundant, deleted repeats of tsLB2 defective virus DNA.

Cell Line↗

PubMed1983

In vivo induction of neoplastic growth in nude mouse connective tissue adjacent to xenografted human tumors.

Induction of neoplastic growth of murine stroma cells within the human tumor xenograft was observed after serial passage of CEA and beta 2-microglobulin producing human colonic SLu tumor xenografts in nu/nu BALB/c mice. Mouse tumors within the human tumor xenografts were identified using specific immunohistologic staining techniques for mouse histocompatibility marker or human CEA. These mixed tumors could be distinguished from normal human tumor xenografts by a different relationship between development of the tumor marker in the serum and tumor size. We were able to establish transformed murine cells from human xenografts, either induced by SC injection of 1 X 10(6) tumor cells of the SLu cell line or by human SLu or mammary carcinoma tissue serially passaged in athymic animals. The established human and murine cell lines were characterized by cytogenetic methods. Transformed murine cells were then continuously passaged in tissue culture. The transformed mouse fibroblasts proved to possess tumorigenicity in nude mice. In the case of SLu-derived mouse tumor cells, tumors also developed in the immunocompetent BALB/c mice using 1 X 10(6) to 5 X 10(6) tumor cells for SC transplantation.

Adenocarcinoma↗

PubMedDate not supplied

The isolation of an immortalized and tumorigenic cell line from p21WAF1 null mouse bladders.

Given a role for the deregulation of p21WAF1 in the progression of bladder tumors, we examined the growth of cultured urothelial cells from wild-type and p21WAF1 null bladders. Bladders were excised, minced from euthanized p21WAF1 and wild-type mice, treated overnight with dispase, and then placed into flasks coated with collagen type I in Dulbecco modified Eagle medium with 10% fetal calf serum. After an overnight incubation, the media was replaced with a serum-free media and a portion of explants were treated with 12-O-tetrade-canoylphorbol-13-acetate (TPA) on day 7 and continued for either 4 or 9 wk. The urothelial origin of any surviving epithelial cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) using uroplakin II-specific primers, and the expression of the cell cycle-related proteins, p16INK4 and p19ARF, was examined by semiquantitative RT-PCR and Western blotting. Isolated wild-type and serially passaged p21WAF1 null epithelial-like cells were then injected subcutaneously into nude mice. We found that phorbol ester treatment at two different concentrations significantly enhanced uroepithelial colony formation from isolated wild-type mouse bladder tissue. On the other hand, significantly fewer urothelial colonies were derived from p21WAF1 null bladder cells treated with phorbol ester. Although there was apparent senescence and cell death of epithelial foci and stromal cells in phorbol ester-treated and -untreated p21WAF1 null cultures, after 3 mo there was an apparent subpopulation of epitheloid cells that overgrew each flask. There was a significant decrease in the number of these serially passaged cells in the G1 phase of the cell cycle when compared with initial explant wild-type or p21WAF1 null cells. This subpopulation of epitheloid cells expressed the mouse uroplakin II gene, indicating a urothelial phenotype, but did not express either the p16INKa or p19ARF proteins, whereas p21WAF1 null bladders express both proteins. There was also a high level of expression of the p53 protein and a significant decrease in the expression of the p19ARF transcript in both p21WAF1 null bladder and p21WAF1 null cells. These p21WAF1 null cells could be easily passaged and when injected subcutaneously into nude mice, large tumors developed. Therefore, it appears that a subpopulation of urothelial cells from the p21WAF1 null bladder can develop a tumorigenic phenotype in vitro.

Animals↗

PubMed1996

Long-circulating bacteriophage as antibacterial agents.

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

Animals↗

PubMed2004

Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa.

OBJECTIVES: Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNA gyrase/topoisomerase IV, or cause activation of various efflux systems. We have analysed the effect of quinolone resistance caused by DNA gyrase/topoisomerase IV mutations on bacterial fitness. METHODS: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified. Mutant fitness was determined by measuring growth rates in vitro. Mutants with reduced growth rates were serially passaged to obtain growth-compensated mutants. The level of DNA supercoiling was determined by isolating plasmid DNA from the susceptible, resistant and compensated mutants and comparing the topoisomer distribution patterns by gel electrophoresis in the presence of chloroquine. RESULTS: Low-level resistance (4-48 mg/L) was caused by single mutations in gyrA or gyrB. Among these strains, three out of eight mutants showed lower fitness, whereas high-level resistant (>256 mg/L) mutants with double mutations in gyrA and parC, parE, nfxB or unknown genes all showed a reduced fitness. Slow-growing resistant mutants with a gyrA mutation had decreased DNA supercoiling. After serial passage in laboratory medium, mutant fitness was increased by compensatory mutation(s) that restored supercoiling to normal levels. The compensatory mutation(s) was not located in any of the genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) that were expected to affect supercoiling. CONCLUSIONS: Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P. aeruginosa.

Anti-Infective Agents↗

PubMed2002

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses.

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.

Animals↗

PubMed1966

L form of Neisseria gonorrhoeae.

Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609-1614. 1966.-L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO(2) tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium.

Anti-Bacterial Agents↗

PubMed1981

Establishment and maintenance of a human glioma transplanted serially to hereditary asplenic-athymic (lasat) mice.

A tumor line in asplenic-athymic (lasat) mice designated KNS-GL-2 was established from a recurrent anaplastic astrocytoma of the cerebrum in a 15-year-old boy (NS761072). The tumor line increased in terms of the rate of tumor take and growth with serial passage, forming a nodular appearance and massive intratumoral hemorrhage at the third generation. Microscopically, tumors developed in lasat mice wee similar to the original tumor and consisted of cells with round and oval nuclei and a clear cytoplasm, which was positive for S-100 and GFA proteins by the immunoperoxidase method. Large clusters of tumor cells with dark scanty cytoplasm seen at the third generation lost their astrocytic appearance and were negative for both S-100 and GFA proteins. This was considered to be evidence for dedifferentiation or malignant transformation. There was a scattering of tumor cells with clear cytoplasm in these clusters, with a positive reaction to S-100 protein. It was possible that tumor cells dedifferentiated during serial passage but still retained the potential to differentiate into astrocytes.

Animals↗

PubMed1952

Acute hepatitis associated with mouse leukemia. I. Pathological features and transmission of the disease.

On four occasions a naturally occurring mouse leukemia, which was maintained by serial passage in weanlings of the Princeton strain, was superseded by a syndrome typical of acute hepatitis. Once initiated, the disease was regularly transmissible by the injection of liver suspensions of sick mice. It was also passed, though irregularly, by feeding such suspensions, and it also followed cannibalism. The course of the disease after intraperitoneal injection of liver suspensions into normal weanlings was commonly less than 7 days and the mortality rate nearly 100 per cent. Focal or diffuse necrosis of the liver was the only constant lesion at autopsy. On recovery, which occurred only in exceptional cases, cirrhosis was often found. The primary source of the disease was undetermined. Latent carriage by healthy mice was not detectable on direct examination nor by the serial passage of suspensions of normal livers.

Acute Disease↗

PubMed1992

Strain-specific selection of genome segments in avian reovirus coinfections.

To determine whether selection of genome segments in coinfections is strain-specific, chicken embryo fibroblasts were coinfected with avian reovirus strain 883 and one of three other avian reovirus strains (176, S1133 and 81-5). Viral progeny from each coinfection (883 x 176, 883 x S1133 or 883 x 81-5) was serially passaged at a low m.o.i. The electropherotypes of the coinfection progeny and those of the plaque-derived clones obtained from passages 1 and 20 were analysed. Two 883 segments (M2 and S2) were found to be selected in the 883 x 176 coinfection, three 883 segments (M2, M3 and S2) in the 883 x S1133 coinfection, and only one 883 segment (M3) in the 883 x 81-5 coinfection, i.e. different 883 genome segments were selected in the three coinfections. It was, therefore, concluded that selection of genome segments in a coinfection of a given cell line is virus strain-specific. The selection of genome segments in coinfections was shown to be due to enhanced infectivity of the reassortants that were formed in the coinfections. In addition, defective interfering particles that lack the S1 segment were identified in the 883 x 81-5 coinfection progeny following serial passage. Selection of genome segment(s) in coinfections as described herein may have potential importance on the effect and production of divalent or multivalent vaccines.

Animals↗